Therapy-related myelodysplastic syndrome (tMDS) is a severe form of MDS caused by exposure to chemotherapeutics, particularly alkylators, used as treatment for other malignancies. tMDS makes up 10 to 20% of new MDS cases and the incidence is rising. Only a small percentage of patients exposed to alkylators develop tMDS and little is known about what predisposes patients to disease. Current evidence suggests a genetic component to predisposition. Mutations in genes associated with the DNA damage response and apoptosis have been implicated in predisposition to some therapy-related malignancies. However, aside from mutations in a few well known cancer predisposition syndromes, genetic alterations predisposing to tMDS are unknown. Identification of genetic variants that predispose to tMDS could allow stratification of patients at risk to better prevent or manage the disease. Our lab has found that susceptibility to tMDS is a heritable trait in mice, with some inbred strains being susceptible and others resistant. Preliminary data shows that altered expression of genes of the intrinsic apoptotic cascade correlates with susceptibility in this model. Furthermore, hematopoietic stem/progenitor cells (HSPCs) from susceptible strains underwent apoptosis at a lower rate than their resistant counterparts after treatment with alkylators. Thus, we hypothesize that reduced function of the intrinsic apoptotic cascade predisposes to tMDS. To test our hypothesis, we will analyze three genetic models of apoptotic deficiency in mice: loss of Caspase-9 (Casp9) or Apaf1 or overexpression of Bcl2. In Aim 1 we will take an in vitro approach to determine the functional consequences of defective apoptosis after genotoxic stress. Defective apoptosis could allow cells to survive with excessive DNA damage, giving rise to a cell population at risk for dysplasia. To test this idea, we will expose cells to N-ethyl-N-nitrosourea (ENU), an alkylator, and measure apoptosis and DNA damage. We will study fetal liver cells (FLCs) from Casp9 and Apaf1 deficient mice and adult hematopoietic cells from mice overexpressing Bcl2 (Vav-Bcl2 strain). In Aim 2, we will examine the effects of defective apoptosis on the clonogenic potential of HSPCs in vitro and in vivo. MDS is a clonal disease even in its early stages and HSPCs are the cells most likely to give rise to these dysplastic clones. We believe that defective apoptosis will affect the colony forming potential of HSPCs after alkylator treatment, resulting in the growth of a clonal population of cells that could cause disease. To determine how alterations in apoptosis impact HSPC clonogenic potential, we will perform colony forming assays in vitro and competitive repopulation assays in vivo on wild type and mutant cells. In Aim 3, we will examine the effect of defective apoptosis on the rate of MDS in mice. To determine if defects in apoptosis confer susceptibility to tMDS, we will transplant irradiated mice with bone marrow or FLCs with one of our mutations, with or without ENU treatment, and examine at three and nine months post-transplant for signs of MDS, including cytopenias and dysplastic erythroid and/or myeloid cells in the bone marrow.